小麦TaHTAS-5A基因的克隆、表达分析及亚细胞定位
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摘要
非生物胁迫对小麦的生长发育具有不良影响,克隆与非生物胁迫相关的基因,并对其结构及表达特性进行分析,可以为进一步探索小麦的抗逆机制和抗逆育种奠定基础。本研究利用同源克隆技术从普通小麦品种中国春中克隆了水稻E3泛素连接酶基因OsHTAS的同源基因TaHTAS-5A(GeneBank登录号:MF967573),并利用生物信息学方法对其基因序列特征进行分析;利用qRT-PCR技术对该基因在小麦不同组织及不同逆境胁迫下的表达模式进行分析;同时,对该基因的表达产物进行了亚细胞定位。结果表明,小麦TaHTAS-5A基因含有220bp的5′UTR、1 242bp的ORF以及126bp的3′UTR,共编码413个氨基酸;基因组分析表明,该基因全长3 819bp,共包含4个外显子和3个内含子;缺体-四体定位表明该基因位于小麦5A染色体上;蛋白结构分析显示,TaHTAS-5A蛋白的N端包含四个跨膜结构域,C端包含一个E3泛素连接酶特有的RING finger保守结构域,具有植物E3泛素连接酶的结构特征;qRT-PCR结果显示,TaHTAS-5A对高温、低温和ABA都有响应,对干旱和盐响应不敏感;TaHTAS-5A在小麦的根、茎、叶和幼穗等不同组织中均有表达,但表达量有差异,在叶和穗中的表达量明显高于根部和茎部;亚细胞定位结果表明,TaHTAS-5A位于细胞膜上。本研究为进一步探究小麦TaHTAS-5A基因的功能奠定了基础,也为小麦非生物胁迫抗性改良提供了一定的理论依据。
Abiotic stress has adverse effects on plant growth and development.Cloning the abiotic stress related genes laid the foundation for further exploring the mechanism of plant stress resistance and provided a theoretical basis for the stress resistance breeding of wheat through the analysis of the gene structure and expression of the target gene.In this study,we cloned the gene TaHTAS-5 A(GeneBank accession number:MF967573)homologous to rice E3 ubiquitin ligase gene OsHTASfrom Chinese spring in Triticum aestivum.We analyzed its sequence features by bioinformatic techniques.The expression pattern of TaHTAS-5 Ain different tissues and under different stresses of wheat was analyzed by qRT-PCR.At the same time,the expression products of this gene were analyzed by subcellular localization.The result show that the TaHTAS-5 A gene contained 220 bp 5′UTR,1 242 bp intact CDS and 126 bp 3′UTR,encoding 413 amino acids.Genomic analysis showed that the full length of TaHTAS-5 Ain genome is 3819 bp,containing four exons and three introns.Nullisomic lo-calization indicated that the gene is located on wheat 5 A chromosome.Protein structure analysis showed that the N-terminus of TaHTAS-5 Aprotein included four transmembrane domains and C-terminal contained an E3 ubiquitin ligase-specific conservative RING finger domain,which has the structural characteristics of plant E3 ubiquitin ligase.qRT-PCR results demonstrated that TaHTAS-5 A was responsive to high temperature,low temperature and ABA,but insensitive to drought and salt stress.Tissue expression analysis showed that TaHTAS-5 A expressed in roots,stems,leaves and young spike,where it was least expressed in root and stem,but highly expressed in leaves and spikes.Subcellular localization showed that TaHTAS-5 Ais located on the cell membrane.The results laid a foundation for further exploring the function of wheat TaHTAS-5 A gene and provided some theoretical basis for improving resistance to abiotic stress in wheat.
Abiotic stress has adverse effects on plant growth and development.Cloning the abiotic stress related genes laid the foundation for further exploring the mechanism of plant stress resistance and provided a theoretical basis for the stress resistance breeding of wheat through the analysis of the gene structure and expression of the target gene.In this study,we cloned the gene TaHTAS-5 A(GeneBank accession number:MF967573)homologous to rice E3 ubiquitin ligase gene OsHTASfrom Chinese spring in Triticum aestivum.We analyzed its sequence features by bioinformatic techniques.The expression pattern of TaHTAS-5 Ain different tissues and under different stresses of wheat was analyzed by qRT-PCR.At the same time,the expression products of this gene were analyzed by subcellular localization.The result show that the TaHTAS-5 A gene contained 220 bp 5′UTR,1 242 bp intact CDS and 126 bp 3′UTR,encoding 413 amino acids.Genomic analysis showed that the full length of TaHTAS-5 Ain genome is 3819 bp,containing four exons and three introns.Nullisomic lo-calization indicated that the gene is located on wheat 5 A chromosome.Protein structure analysis showed that the N-terminus of TaHTAS-5 Aprotein included four transmembrane domains and C-terminal contained an E3 ubiquitin ligase-specific conservative RING finger domain,which has the structural characteristics of plant E3 ubiquitin ligase.qRT-PCR results demonstrated that TaHTAS-5 A was responsive to high temperature,low temperature and ABA,but insensitive to drought and salt stress.Tissue expression analysis showed that TaHTAS-5 A expressed in roots,stems,leaves and young spike,where it was least expressed in root and stem,but highly expressed in leaves and spikes.Subcellular localization showed that TaHTAS-5 Ais located on the cell membrane.The results laid a foundation for further exploring the function of wheat TaHTAS-5 A gene and provided some theoretical basis for improving resistance to abiotic stress in wheat.
引文
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[23]HUNTER T.A thousand and one protein kinases[J].Cell,1987,50(6):823.
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[25]KATAYA A R,SCHEI E,LILLO C.Towards understanding peroxisomal phosphoregulation in Arabidopsis thaliana[J].Planta,2016,243(3):699.
[26]郭丽香,高世庆,唐益苗,等.小麦TaCRF2基因的克隆及其在烟草中的初步功能验证[J].作物学报,37(8):1389.GUO L X,GAO S Q,TANG Y M,et al.TaCRF2 Gene Isolation from Triticum aestivum and primary function validation in tobacco(Nicotiana tabacum L)[J].ActaAgronomica Sinica,2011,37(8):1389.
[2]刘诗航,王彩香,毛新国,等.小麦蛋白磷酸酶2A调节亚基基因TaBβ-11的克隆及其在非生物胁迫下的表达特性[J].中国农业科学,2010,43(11):2197.LIU S H,WANG C X,MAO X G,et al.Cloning of protein phosphatase 2Aregulatory subunit gene TaBβ-1 and its expression patterns under abiotic stresses in wheat[J].ScientiaAgricultura Sinica,2010,43(11):2197.
[3]TUTEJA N,TIBURCIO A F,FORTES A M,et al.Plant abiotic stress.Introduction to PSB special issue[J].Plant Signaling and Behavior,2011,6(2):173.
[4]HIRAYAMA T,SHINOZAKI K.Research on plant abiotic stress responses in the post-genome era:Past,present and future[J].Plant Journal,2010,61(6):1041.
[5]ZHU J K.Salt and drought stress signal transduction in plants[J].Annual Review of Plant Biology,2002,53(53):247.
[6]QIN F,SHINOZAKI K,YAMAGUCHI-SHINOZAKIA K.Achievements and challenges in understanding plant abiotic stress responses and tolerance[J].Plant Cell Physiology,2011,52(9):1569.
[7]AGARWAL M,HAO Y,KAPOOR A,et al.A R2R3type MYB transcription factor is involved in the cold regulation of CBF genes and in acquired freezing tolerance[J].Journal of Biological Chemistry,2006,281(49):37636.
[8]SAKUMAY,MARUYAMA K,OSAKABE Y,et al.Functional analysis of an Arabidopsis transcription factor,DREB2A,involved in drought-responsive gene expression[J].Plant Cell,2006,18(5):1292.
[9]RAY S,DANASANA P K,GIRI J,et al.Modulation of transcription factor and metabolic pathway genes in response to water-deficit stress in rice[J].Functional and Integrative Genomics,2011,11(1):157.
[10]CUI F,LIU L,ZHAO Q,et al.Arabidopsis ubiquitin conjugase UBC32is an ERAD component that functions in brassinosteroid-mediated salt stress tolerance[J].Plant Cell,2012,24(1):233.
[11]KIM J H,KIM W T.The Arabidopsis RING E3ubiquitin ligase AtAIRP3/LOG2participates in positive regulation of high-salt and drought stress responses[J].Plant Physiology,2013,162:1733.
[12]KOOPS P,PELSER S,IGNATZ M,et al.EDL3is an F-box protein involved in the regulation of abscisic acid signalling in Arabidopsis thaliana[J].Journal ofExperimental Botany,2011,62(15):5547.
[13]CLARKE D J,MONDESERT G,SEGAL M,et al.Dosage suppressors of pds1implicate ubiquitin-associated domains in checkpoint control[J].Molecular and Cellular Biology,2001,21(6):1997.
[14]GWIZDEK C,IGLESIAS N,RODRIGUZE M S,et al.Ubiquitin-associated domain of Mex67synchronizes recruitment of the mRNA export machinery with transcription[J].Proceedings of the National Academy of Sciences of the United States of America,2006,103(44):16376.
[15]DREHER K,CALLIS J.Ubiquitin,hormones and biotic stress in plants[J].Annals of Botany,2007,99:787.
[16]VIERSTRA R D.The ubiquitin-26Sproteasome system at the nexus of plant biology[J].Nature Reviews Molecular Cell Biology,2009,10(6):385.
[17]SADANANDOM A,BAILEY M,EWAIN R,et al.The ubiquitin-proteasome system:Central modifier of plant signalling[J].New Phytologist,2012,196(1):13.
[18]RYU M Y,CHO S K,KIM W T.The Arabidopsis C3H2C3-Type RING E3ubiquitin ligase AtAIRP1is a positive regulator of an abscisic acid-dependent response to drought stress[J].Plant Physiology,2011,154(4):1983.
[19]NING Y S,JANTASURIYARAT C,ZHAO Q,et al.The SINA E3ligase OsDIS1 negatively regulates drought response in rice[J].Plant Physiology,2011,157(1):242.
[20]LIU J,ZHANG C,WEI C,et al.The RING Finger Ubiquitin E3Ligase OsHTAS enhances heat tolerance by promoting H2O2-induced stomatal closure in rice[J].Plant Physiology,2016,170(1):429.
[21]WEI H,LIU J,WANG Y,et al.A dominant major locus in chromosome 9of rice(Oryza sativa L.)confers tolerance to48℃high temperature at seedling stage[J].Journal of Heredity,2013,104(2):287.
[22]耿晓丽,臧新山,王飞,等.小麦耐热相关转录因子基因TabZIP28的分离及功能分析[J].农业生物技术学报,2016,24(2):157.GENG X L,ZANG X S,WANG F,et al.Isolation and function analysis of heat stress related transcription factor gene TabZIP28in wheat(Triticum aestivum)[J].Journal of Agricultural Biotechnology,2016,24(2):157.
[23]HUNTER T.A thousand and one protein kinases[J].Cell,1987,50(6):823.
[24]ZHANG S,KLESSIG D F.MAPK cascades in plant defense signaling[J].Trends in Plant Science,2001,6(11):520.
[25]KATAYA A R,SCHEI E,LILLO C.Towards understanding peroxisomal phosphoregulation in Arabidopsis thaliana[J].Planta,2016,243(3):699.
[26]郭丽香,高世庆,唐益苗,等.小麦TaCRF2基因的克隆及其在烟草中的初步功能验证[J].作物学报,37(8):1389.GUO L X,GAO S Q,TANG Y M,et al.TaCRF2 Gene Isolation from Triticum aestivum and primary function validation in tobacco(Nicotiana tabacum L)[J].ActaAgronomica Sinica,2011,37(8):1389.